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Abstract:

A nucleic acid probe for classification of pathogenic bacterial species is
capable of collectively detecting bacterial strains of the same species
and differentially detecting them from other bacterial species. Any one
of the base sequence of SEQ ID NO. 94 and complementary or modified
sequences thereof or a combination of at least two of them is used for
detecting the gene of an infectious disease pathogenic bacterium.

Claims:

1. A probe for detecting a gene of infectious disease pathogenic
bacterium, Propionibacterium (genus), having any one of the following
base sequences (1) to (2):(1) GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94)
or a complementary sequence thereof;(2) a modified sequence prepared such
that any one of the sequences of SEQ ID NO. 94 and the complementary
sequence thereof is subjected to base deletion, substitution, or addition
as far as the modified sequence retains a function as the probe.

2. A probe-immobilized carrier, wherein at least one first probe according
to claim 1 is arranged on a solid-phase carrier.

3. A probe-immobilized carrier according to claim 2, wherein the
probe-immobilized carrier comprises at least one second probe having any
one of the base sequences of SEQ ID NOS. 35 to 94 as mentioned in the
specification immobilized at a position spaced from the first probe.

4. A kit for detecting a gene of Propionibacterium (genus), comprising:a
probe according to claim 1; anda reagent for detecting a reaction between
the probe and a target nucleic acid.

5. A kit according to claim 4, wherein the reagent contains a primer for
amplifying the gene of Propionibacterium (genus), and the primer
includes:(5) an oligonucleotide having a base sequence of 5'
gcggcgtgcttaacacatgcaag 3' (SEQ ID NO: 5); and(25) an oligonucleotide
having a base sequence of 5' atccagccgcaccttccggtac 3' (SEQ ID NO: 25).

6. A gene detection kit, comprising:a probe-immobilized carrier according
to claim 3; anda reagent for detecting a reaction between the probes and
a target nucleic acid,wherein the reagent contains a primer including at
least one oligonucleotide selected from the following items (1) to (21)
and at least one oligonucleotide selected from the following items (22)
to (28):(1) an oligonucleotide having a base sequence of 5'
gcggcgtgcctaatacatgcaag 3' (SEQ ID NO: 1);(2) an oligonucleotide having a
base sequence of 5' gcggcaggcctaacacatgcaag 3' (SEQ ID NO: 2);(3) an
oligonucleotide having a base sequence of 5' gcggcaggcttaacacatgcaag 3'
(SEQ ID NO: 3);(4) an oligonucleotide having a base sequence of 5'
gcggtaggcctaacacatgcaag 3' (SEQ ID NO: 4);(5) an oligonucleotide having a
base sequence of 5' gcggcgtgcttaacacatgcaag 3' (SEQ ID NO: 5);(6) an
oligonucleotide having a base sequence of 5' gcgggatgccttacacatgcaag 3'
(SEQ ID NO: 6);(7) an oligonucleotide having a base sequence of 5'
gcggcatgccttacacatgcaag 3' (SEQ ID NO: 7);(8) an oligonucleotide having a
base sequence of 5' gcggcatgcttaacacatgcaag 3' (SEQ ID NO: 8);(9) an
oligonucleotide having a base sequence of 5' gcggcgtgcttaatacatgcaag 3'
(SEQ ID NO: 9);(10) an oligonucleotide having a base sequence of 5'
gcggcaggcctaatacatgcaag 3' (SEQ ID NO: 10);(11) an oligonucleotide having
a base sequence of 5' gcgggatgctttacacatgcaag 3' (SEQ ID NO: 11);(12) an
oligonucleotide having a base sequence of 5' gcggcgtgcctaacacatgcaag 3'
(SEQ ID NO: 12);(13) an oligonucleotide having a base sequence of 5'
gcggcgtgcataacacatgcaag 3' (SEQ ID NO: 13);(14) an oligonucleotide having
a base sequence of 5' gcggcatgcctaacacatgcaag 3' (SEQ ID NO: 14);(15) an
oligonucleotide having a base sequence of 5' gcggcgcgcctaacacatgcaag 3'
(SEQ ID NO: 15);(16) an oligonucleotide having a base sequence of 5'
gcggcgcgcttaacacatgcaag 3' (SEQ ID NO: 16);(17) an oligonucleotide having
a base sequence of 5' gcgtcatgcctaacacatgcaag 3' (SEQ ID NO: 17);(18) an
oligonucleotide having a base sequence of 5' gcgataggcttaacacatgcaag 3'
(SEQ ID NO: 18);(19) an oligonucleotide having a base sequence of 5'
gcgacaggcttaacacatgcaag 3' (SEQ ID NO: 19);(20) an oligonucleotide having
a base sequence of 5' gctacaggcttaacacatgcaag 3' (SEQ ID NO: 20);(21) an
oligonucleotide having a base sequence of 5' acagaatgcttaacacatgcaag 3'
(SEQ ID NO: 21);(22) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccgatac 3' (SEQ ID NO: 22);(23) an oligonucleotide having
a base sequence of 5' atccaaccgcaggttcccctac 3' (SEQ ID NO: 23);(24) an
oligonucleotide having a base sequence of 5' atccagccgcaggttcccctac 3'
(SEQ ID NO: 24);(25) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccggtac 3' (SEQ ID NO: 25);(26) an oligonucleotide having
a base sequence of 5' atccagcgccaggttcccctag 3' (SEQ ID NO: 26);(27) an
oligonucleotide having a base sequence of 5' atccagccgcaggttctcctac 3'
(SEQ ID NO: 27); and(28) an oligonucleotide having a base sequence of 5'
atccagccgcacgttcccgtac 3' (SEQ ID NO: 28).

7. A probe set for detecting a gene of infectious disease pathogenic
bacterium, Propionibacterium (genus), including at least two probes
selected from the following items (A) to (D):(A) a probe having a base
sequence represented by GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94);(B) a
probe having a complementary sequence of the base sequence represented by
SEQ ID NO. 94;(C) a probe having a modified sequence obtained by base
deletion, substitution, or addition on the base sequence represented by
SEQ ID NO. 94 as far as it retains the function of a probe for detecting
the gene of Propionibacterium (genus); and(D) a probe having a modified
sequence obtained by base deletion, substitution, or addition on the
complementary sequence of the base sequence represented by SEQ ID NO. 94
as far as it retains the function of a probe for detecting the gene of
Propionibacterium (genus).

8. A probe-immobilized carrier according to claim 2, wherein the at least
one first probe includes at least two first probes selected from the
following items (A) to (D):(A) a probe having a base sequence represented
by GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94);(B) a probe having a
complementary sequence of the base sequence represented by SEQ ID NO.
94;(C) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ ID NO.
94 as far as it retains the function of a probe for detecting the gene of
Propionibacterium (genus); and(D) a probe having a modified sequence
obtained by base deletion, substitution, or addition on the complementary
sequence of the base sequence represented by SEQ ID NO. 94 as far as it
retains the function of a probe for detecting the gene of
Propionibacterium (genus), and wherein the at least two first probes are
arranged on a solid-phase carrier at intervals from each other.

9. A probe-immobilized carrier according to claim 8, wherein the
probe-immobilized carrier comprises at least one second probe having any
one of the base sequences of SEQ ID NOS. 35 to 94 as mentioned in the
specification immobilized at a position spaced from the at least two
first probes.

10. A method of detecting a gene of Propionibacterium (genus) in an
analyte by using a probe-immobilized carrier, comprising the steps of:(i)
reacting the analyte with a probe-immobilized carrier according to claim
2; and(ii) detecting the presence or absence of a reaction of the probe
on the probe-immobilized carrier with a nucleic acid in the analyte, or
detecting the strength of a hybridization reaction of the probe on the
probe-immobilized carrier with a nucleic acid in the analyte.

11. A method according to claim 10, further comprising the step of
carrying out PCR amplification of the target nucleic acid in the analyte
by using a primer including the following oligonucleotides:(5) an
oligonucleotide having a base sequence of 5' gcggcgtgcttaacacatgcaag 3'
(SEQ ID NO: 5); and(25) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccggtac 3' (SEQ ID NO: 25).

Description:

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002]The present invention relates to a probe and a probe set for
detecting a gene of infectious disease pathogenic bacterium,
Propionibacterium (genus), which are useful for detection and
identification of the causative organism of an infectious disease, a
probe-immobilized carrier on which the probe or the probe set is
immobilized, a genetic testing method using the probe-immobilized
carrier, and a genetic testing kit to be used for the method.

[0003]2. Related Background Art

[0004]Heretofore, reagents for and methods of quickly and accurately
detecting the causative organisms of infectious diseases in analytes have
been proposed. For instance, Japanese Patent Application Laid-Open No.
H08-089254 discloses oligonucleotides having specific base sequences,
which can be respectively used as probes and primers for detecting
pathogenic bacteria of candidiasis and aspergillosis, and a method of
detecting target bacteria using such oligonucleotides. In addition, the
same patent document also discloses a set of primers used for
concurrently amplifying a plurality of target bacteria by PCR. In other
words, those primers are used for the PCR amplification of nucleic acid
fragments from fungi, which serve as a plurality of targets, in an
analyte. Target fungal species in the analyte can be identified by
detecting the presence of a specific part of the sequence by a
hybridization assay using probes specific to the respective fungi and the
nucleic acid fragments amplified by the respective primers.

[0005]On the other hand, the method to use probe array in which probes
having sequences complementary to the respective base sequences are
arranged at intervals on a solid support is known as a method capable of
simultaneously detecting a plurality of oligonucleotides having different
base sequences (Japanese Patent Application Laid-Open No. 2004-313181).

SUMMARY OF THE INVENTION

[0006]However, it is no easy task to design a probe for specifically
detecting a gene of an infectious disease pathogenic bacterium in a
sample. That is, as well as the target gene, the sample may further
contain genes of other infectious disease pathogenic bacteria. Thus, it
is no easy task to design the probe that specifically detects the gene of
the infectious disease pathogenic bacterium while suppressing the cross
contamination which is the influence of the presence of the genes of
other infectious disease pathogenic bacteria. Under such circumstances,
the inventors of the present invention have studied for obtaining a probe
which allows accurate detection of a gene of an infectious disease
pathogenic bacterium as mentioned hereinbelow while maintaining the cross
contamination level low even when a sample in which genes of different
bacteria are present is used. As a result, the inventors of the present
invention have finally found a probe capable of precisely detecting the
gene of the infectious disease pathogenic bacterium, Propionibacterium
(genus).

[0007]A first object of the present invention is to provide a probe and a
probe set, which can precisely identify a gene of a target bacterium from
an analyte in which various bacteria are concurrently present. Another
object of the present invention is to provide a probe-immobilized carrier
which can be used for precisely identifying a target bacterium from an
analyte in which various bacteria are concurrently present. Still another
object of the present invention is to provide a genetic testing method
for detecting a target bacterium, which can quickly and precisely detect
the target bacterium from various bacteria in an analyte when they are
present therein, and a kit for such a method.

[0008]The probe for detecting a gene of infectious disease pathogenic
bacterium, Propionibacterium (genus), of the present invention has any
one of the following base sequences (1) to (3):

(1) GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94) or a complementary
sequence thereof;(2) a modified sequence prepared such that any one of
the sequences of SEQ ID NO. 94 and the complementary sequence thereof is
subjected to base deletion, substitution, or addition as far as the
modified sequence retains a function as the probe.

[0009]In addition, the probe set for detecting a gene of infectious
disease pathogenic bacterium, Propionibacterium (genus), of the present
invention includes at least two probes selected from the following items
(A) to (D):

(A) a probe having a base sequence represented by
GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94);(B) a probe having a
complementary sequence of the base sequence represented by SEQ ID NO.
94;(C) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ ID NO.
94 as far as it retains the function of a probe for detecting the gene of
Propionibacterium (genus);(D) a probe having a modified sequence obtained
by base deletion, substitution, or addition on the complementary sequence
of the base sequence represented by SEQ ID NO. 94 as far as it retains
the function of a probe for detecting the gene of Propionibacterium
(genus); and the characteristic feature of the probe-immobilized carrier
of the present invention is that at least one of the above-mentioned
probes (A) to (D) is immobilized on a solid-phase carrier, and when a
plurality of probes are employed, the respective probes are arranged at
intervals.

[0010]The method of detecting a gene of an infectious disease pathogenic
bacterium, Propionibacterium (genus), in an analyte by using a
probe-immobilized carrier of the present invention includes the steps of:

(i) reacting the analyte with the probe-immobilized carrier having the
above-mentioned constitution; and(ii) detecting the presence or absence
of a reaction of the probe on the probe-immobilized carrier with a
nucleic acid in the analyte, or detecting the strength of a hybridization
reaction of the probe on the probe-immobilized carrier with a nucleic
acid in the analyte.

[0011]The characteristic feature of the kit for detecting an infectious
disease pathogenic bacterium, Propionibacterium (genus), of the present
invention is to include at least one of the above-mentioned probes (A) to
(D), and a reagent for detecting a reaction between the probe and a
target nucleic acid.

[0012]According to the present invention, when an analyte is infected with
the above-mentioned causative bacterium, the bacterium can be more
quickly and precisely identified from the analyte even if the analyte is
simultaneously and complexly infected with other bacteria in addition to
the above-mentioned bacterium. In particular, Propionibacterium (genus)
can be detected while precisely distinguishing it from Escherichia coli
which may otherwise cause cross contamination.

[0013]Further features of the present invention will become apparent from
the following description of exemplary embodiments with reference to the
attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a diagram illustrating a 1st PCR protocol.

[0015]FIG. 2 is a diagram illustrating a 2nd PCR protocol.

DETAILED DESCRIPTION OF THE INVENTION

[0016]The inventors of the present invention have obtained almost all of
bacteria (represented by (1) to (80) below), which have been known as
septicemia pathogenic bacteria so far, from the respective depository
institutions and identified the 16S rRNA gene sequences of all the
bacteria.

[0017]Subsequently, while making a comparison of all the identified
sequences, probe sequences for Propionibacterium (genus) were
investigated in detail and the probes of the present invention, which can
identify Propionibacterium (genus), have finally been found out.

[0018]The deposition numbers of the bacterial species obtained are shown
in the respective parentheses on the right side in the above. Bacterial
species having deposition numbers beginning with "ATCC", "JCM" and "NBRC"
are available from American Type Culture Collection, Japan Collection of
Microorganisms (RIKEN BioResource Center) and National Board for
Respiratory Care, respectively.

[0019]The present invention provides an oligonucleotide probe for
identifying an infectious disease pathogenic bacterium (hereinafter,
simply referred to as a probe) and a probe set including a combination of
two or more probes. The use of such a probe or a probe set allows the
detection of the following bacterium which will cause inflammation by
infection.

[0020][Bacterial Name]

[0021]Propionibacterium (Genus)

[0022]That is, the probe of the present invention can detect the 16S rRNA
gene sequence among genes of the above-mentioned bacterium, having the
following sequences:

(A) a probe having a base sequence represented by
GCTTTCGATACGGGTTGACTTGAGGAA (SEQ ID NO. 94);(B) a probe having a
complementary sequence of the base sequence represented by SEQ ID NO.
94;(C) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ ID NO.
94 as far as it retains the function of a probe for detecting the gene of
Propionibacterium (genus);(D) a probe having a modified sequence obtained
by base deletion, substitution, or addition on the complementary sequence
of the base sequence represented by SEQ ID NO. 94 as far as it retains
the function of a probe for detecting the gene of Propionibacterium
(genus); and the probe set can be formed using at least two of those
probes.

[0023]The functions of those probes significantly depend on the
specificity of each probe sequence corresponding to the target nucleic
acid sequence of interest. The specificity of a probe sequence can be
evaluated from the degree of coincidence of bases with the target nucleic
acid sequence and the probe sequence. Further, when a plurality of probes
constitute a probe set, the variance of melting temperatures among the
probes may affect the performance of the probe set.

[0024]For designing a probe sequence, a region showing a high specificity
to a specific bacterial species of interest regardless of any differences
in strain is selected. The region contains three or more bases which are
not coincident with corresponding bases in the sequences of any other
bacterial species. The probe sequence is designed so that the melting
temperature between the probe sequence and the corresponding sequence of
the specific bacterial species of interest will differ by 10° C.
or more from the melting temperatures between the probe sequence and the
corresponding sequences of any other bacterial species. Further, one or
more bases can be deleted or added so that the respective probes
immobilized on a single carrier may have melting temperatures within a
predetermined range.

[0025]The inventors of the present invention found out by experiments that
the hybridization intensity of a probe will not be significantly
attenuated if 80% or more of the base sequence is consecutively
conserved. It can therefore be concluded, from the finding, such that any
sequences modified from the probe sequences disclosed in the
specification will have a sufficient probe function if 80% or more of the
base sequence of the probe is consecutively conserved.

[0026]The above-mentioned modified sequences may include any variation as
far as it does not impair the probe's function, or any variation as far
as it hybridizes with a nucleic acid sequence of interest as a detection
target. Above all, it is desirable to include any variation as far as it
can hybridize with a nucleic acid sequence of interest as a detection
target under stringent conditions. Preferable hybridization conditions
confining the variation include those represented in examples as
described below. Here, the term "detection target" used herein may be one
included in a sample to be used in hybridization, which may be a unique
base sequence to the infectious disease pathogenic bacterium, or may be a
complementary sequence to the unique sequence. Further, the variation may
be a modified sequence obtained by deletion, substitution, or addition of
at least one base as far as it retains a function as the probe.

[0027]Those probe sequences are only specific to the DNA sequence coding
for the 16S rRNA of the above-mentioned bacterium, so sufficient
hybridization sensitivity to the sequence will be expected even under
stringent conditions. In addition, any of those probe sequences forms a
stable hybridized product through a hybridization reaction thereof with a
target analyte even when the probe sequences are immobilized on a
carrier, which is designed to produce an excellent result.

[0028]Further, a probe-immobilized carrier (e.g., DNA chip), on which the
probe for detecting the infectious disease pathogenic bacterium of the
present invention, can be obtained by supplying the probe on a
predetermined position on the carrier and immobilizing the probe thereon.
Various methods can be used for supplying the probe to the carrier. Among
them, for example, a method, which can be suitably used, is to keep a
surface state capable of immobilizing the probe on the carrier through a
chemical bonding (e.g., covalent bonding) and a liquid containing the
probe is then provided on a predetermined position by an inkjet method.
Such a method allows the probe to be hardly detached from the carrier and
exerts an additional effect of improving the sensitivity. In other words,
when a stamping method conventionally used and called the Stanford method
is employed to make a DNA chip, the resultant DNA chip has a disadvantage
such that the applied DNA tends to be peeled off. Another one of the
methods of forming DNA chips is to carry out the arrangement of probes by
the synthesis of DNA on the surface of a carrier (e.g., DNA chip from
Affymetrix Co., Ltd.). In such a method of synthesizing probes on a
carrier, it is difficult to make equal the amount of synthesized DNA for
each probe sequence. Thus, the amount of immobilized probe per
immobilization area (spot) for each probe tends to vary considerably.
Such variations in amounts of the respective immobilized probes may cause
incorrect evaluation on the results of the detection with those probes.
Based on this fact, the probe carrier of the present invention is
preferably prepared using the above-mentioned inkjet method. The inkjet
method as described above has an advantage such that the probe can be
stably immobilized on the carrier and hardly detaching from the carrier
to efficiently provide a probe carrier which can carry out detection with
high sensitivity and high accuracy.

[0029]In addition, a probe set may include at least two selected from the
group consisting of SEQ ID NO. 94 as described above and the
complementary sequence thereof and sequences obtained by base deletion,
substitution, or addition on those sequences as far as they retain the
function of a probe for detecting the gene of Propionibacterium (genus).
In this case, the accuracy of detecting the Propionibacterium (genus)
gene can be further improved.

[0030]Hereinafter, preferred embodiments of the present invention will be
described in detail.

[0031]Test objects to be tested using probe carriers (e.g., DNA chips) in
which the probes of the present invention are immobilized on carriers
include those originated from humans and animals such as domestic
animals. For example, a test object is any of those which may contain
bacteria, including: any body fluids such as blood, cerebrospinal fluid,
expectorated sputum, gastric juice, vaginal discharge, and oral mucosal
fluid; and excretions such as urine and feces. All media, which can be
contaminated with bacteria, can be also subjected to a test using a DNA
chip. Such media include: food, drink water and water in the natural
environment such as hot spring water, which may cause food poisoning by
contamination; filters of air cleaners and the like; and so on. Animals
and plants, which should be quarantined in import/export, are also used
as analytes of interest.

[0032]When the sample as described above can be directly used in reaction
with the DNA chip, it is used as an analyte to react with the DNA chip
and the result of the reaction is then analyzed. Alternatively, when the
sample cannot be directly reacted with the DNA chip, the sample was
subjected to extraction, purification, and other procedures for obtaining
a target substance if required and then provided as an analyte to carry
out a reaction with the DNA chip. For instance, when the sample contains
a target nucleic acid, an extract, which may be assumed to contain such a
target nucleic acid, is prepared from a sample, and then washed, diluted,
or the like to obtain an analyte solution followed by reaction with the
DNA chip. Further, as a target nucleic acid is included in an analyte
obtained by carrying out various amplification procedures such as PCR
amplification, the target nucleic acid may be amplified and then reacted
with a DNA chip. Such analytes of amplified nucleic acids include the
following ones:

(a) An amplified analyte prepared by using a PCR-reaction primer designed
for detecting 16S rRNA gene.(b) An amplified analyte prepared by an
additional PCR reaction or the like from a PCR-amplified product.(c) An
analyte prepared by an amplification method other than PCR.(d) An analyte
labeled for visualization by any of various labeling methods.

[0033]Further, a carrier used for preparing a probe-immobilized carrier,
such as a DNA chip, may be any of those that satisfy the property of
carrying out a solid phase/liquid phase reaction of interest. Examples of
the carrier include: flat substrates such as a glass substrate, a plastic
substrate, and a silicon wafer; a three-dimensional structure having an
irregular surface; and a spherical body such as a bead, and rod-, cord-,
and thread-shaped structures. The surface of the carrier may be processed
such that a probe can be immobilized thereon. Especially, a carrier
prepared by introducing a functional group to its surface to enable
chemical reaction has a preferable form from the viewpoint of
reproducibility because the probe is stably bonded in the process of
hybridization reaction.

[0034]Various methods can be employed for the immobilization of probes. An
example of such a method is to use a combination of a maleimide group and
a thiol (--SH) group. In this method, a thiol (--SH) group is bonded to
the terminal of a probe, and a process is executed in advance to make the
carrier (solid) surface have a maleimide group. Accordingly, the thiol
group of the probe supplied to the carrier surface reacts with the
maleimide group on the carrier surface to form a covalent bond, whereby
the probe is immobilized.

[0035]Introduction of the maleimide group can utilize a process of firstly
allowing a reaction between a glass substrate and an aminosilane coupling
agent and then introducing a maleimide group onto the glass substrate by
a reaction of the amino group with an EMCS reagent
(N-(6-maleimidocaproyloxy)succinimide, available from Dojindo).
Introduction of the thiol group to a DNA can be carried out using
5'-Thiol-Modifier C6 (available from Glen Research) when the DNA is
synthesized by an automatic DNA synthesizer.

[0036]Instead of the above-described combination of a thiol group and a
maleimide group, a combination of, e.g., an epoxy group (on the solid
phase) and an amino group (nucleic acid probe terminal), can also be used
as a combination of functional groups to be used for immobilization.
Surface treatments using various kinds of silane coupling agents are also
effective. A probe in which a functional group which can react with a
functional group introduced by a silane coupling agent is introduced is
used. A method of applying a resin having a functional group can also be
used.

[0037]The detection of the gene of the infectious disease pathogenic
bacterium by using the probe-immobilized carrier of the present invention
can be carried out by a genetic testing method including the steps of:

(i) reacting an analyte with a probe-immobilized carrier on which the
probe of the present invention is immobilized;(ii) detecting the presence
or absence of the reaction of a nucleic acid in the analyte with the
probe on the probe-immobilized carrier, or detecting the strength of the
hybridization reaction of a nucleic acid in the analyte with the probe on
the probe-immobilized carrier; and(iii) specifying the probe having
reacted with the nucleic acid in the analyte when the reaction of the
probe with the nucleic acid in the analyte is detected and specifying the
gene of the infectious disease pathogenic bacterium in the analyte based
on the nucleic acid sequence of the probe.

[0038]The probe to be immobilized on the probe-immobilized carrier is at
least one of the above-mentioned items (A) to (D). On the carrier, probes
for detecting bacterial species other than Propionibacterium (genus) may
be immobilized as other probes, depending on the purpose of test. In this
case, the other probes may be those capable of detecting the bacterial
species other than Propionibacterium (genus) without causing cross
contamination and the use of such probes allows simultaneous detection of
a plurality of bacterial species with high accuracy.

[0039]Further, as described above, when the 16S rRNA gene sequence of an
infectious disease pathogenic bacterium in the analyte is amplified by
PCR and provided as a sample to be reacted with a probe carrier, a primer
set for detecting the infectious disease pathogenic bacterium can be
used. The primer set suitably includes at least one selected from
oligonucleotides represented in the following items (1) to (21) and at
least one selected from oligonucleotides represented in the following
items (22) to (28), more suitably includes all the oligonucleotides
represented in the following items (1) to (28):

[0041]For detecting Propionibacterium (genus), at least such a primer may
be included.

[0042]The utilities of the respective primers (1) to (28) for
amplification of Propionibacterium (genus) can be evaluated and confirmed
by, comparing each sequence of SEQ ID NOs. 1 to 28 with a DNA sequence
including the 16S rRNA coding region of Propionibacterium freudenreichii
(ATCC 6207, SEQ ID NO. 95).

[0043]A kit for detecting the infectious disease pathogenic bacterium can
be constructed using at least a probe as described above and a reagent
for detecting a reaction of the probe with a nucleic acid in an analyte.
The probe in the kit can preferably be provided as a probe-immobilized
carrier as described above. Further, the detection reagent may contain a
label to detect the reaction or a primer for carrying out amplification
as a pre-treatment.

EXAMPLES

[0044]Hereinafter, the present invention will be described in more detail
with reference to examples using probes for detecting an infectious
disease pathogenic bacterium to detect Propionibacterium (genus).

Example 1

[0045]In this example, microorganism detection using 2-step PCR will be
described.

[0046]1. Preparation of Probe DNA

[0047]A nucleic acid sequence shown in Table 1 was designed as probe to be
used for detection of Propionibacterium (genus). Specifically, the
following probe base sequence was selected from the genome part coding
for the 16s rRNA gene of Propionibacterium (genus). This probe base
sequence was designed such that it could have an extremely high
specificity to the bacterium, and a sufficient hybridization sensitivity
could be expected without variance for the probe base sequence. The probe
base sequence need not always completely match with those shown in Table
1. Probes having base lengths of 20 to 30 which include the base sequence
shown in Table 1 can also be used, in addition to the probe having the
base sequence shown in Table 1. However, it should be ensured that the
other portion of the base sequence than the portion shown in Table 1 in
such a probe has no effect on the detection accuracy.

[0048]For each probe having a base sequence shown in Table 1, a thiol
group was introduced, as a functional group to immobilize the probe on a
DNA chip, to the 5' terminal of the nucleic acid after synthesis in
accordance with a conventional method. After introduction of the
functional group, purification and freeze-drying were executed. The
freeze-dried probes for internal standard were stored in a freezer at
-30° C.

[0049]2. Preparation of PCR Primers

[0050]2-1. Preparation of PCR Primers for Analyte Amplification

[0051]As 16S rRNA gene (target gene) amplification PCR primers for
pathogenic bacterium detection, nucleic acid sequences shown in Table 2
below were designed. Specifically, primer sets which specifically amplify
the genome parts coding the 16S rRNAs, i.e., primers for which the
specific melting points were made uniform as far as possible at the two
end portions of the 16S rRNA coding region of a base length of 1,400 to
1,700 were designed. In order to simultaneously amplify a plurality of
different bacterial species listed in the following items (1) to (80),
mutants, or a plurality of 16S rRNA genes on genomes, a plurality of
kinds of primers were designed. Note that a primer set is not limited to
the primer sets shown in Table 2 as far as the primer set is available in
common to amplify almost the entire lengths of the 16S rRNA genes of the
pathogenic bacteria.

[0052]The primers shown in Table 2 were purified by high performance
liquid chromatography (HPLC) after synthesis. The twenty-one forward
primers and the seven reverse primers were mixed and dissolved in a TE
buffer solution such that each primer concentration had an ultimate
concentration of 10 pmol/μl.

[0053]2-2. Preparation of Labeling PCR Primers

[0054]In a manner similar to the above-mentioned analyte amplification
primers, oligonucleotides having sequences as shown in Table 3 below were
employed as primers for labeling.

[0055]The primers shown in Table 3 were labeled with a fluorescent dye,
Cy3. The primers were purified by high performance liquid chromatography
(HPLC) after synthesis. The six labeled primers were mixed and dissolved
in a TE buffer solution such that each primer concentration had an
ultimate concentration of 10 pmol/μl.

[0058]First, three species of Propionibacterium (genus), Propionibacterium
acnes (JCM 6473), Propionibacterium avidum (ATCC 25577) and
Propionibacterium freudenreichii (ATCC 6207), were cultured in accordance
with the conventional method. This microbial culture medium was subjected
to the extraction and purification of genome DNA by using a nucleic acid
purification kit (FastPrep FP100A FastDNA Kit, manufactured by Funakoshi
Co., Ltd.).

[0059]3-2. Test of Collected Genome DNA

[0060]The collected genome DNA of the microorganism, Propionibacterium
(genus), was subjected to agarose electrophoresis and 260/280-nm
absorbance determination in accordance with the conventional method.
Thus, the quality (the admixture amount of low molecular nucleic acid and
the degree of decomposition) and the collection amount were tested. In
this example, about 10 μg of the genome DNA was collected. No
degradation of genome DNA or contamination of rRNA was observed. The
collected genome DNA was dissolved in a TE buffer solution at an ultimate
concentration of 50 ng/μl and used in the following experiments.

[0061]4. Preparation of DNA Chip

[0062]4-1. Cleaning of Glass Substrate

[0063]A glass substrate (size: 25 mm×75 mm×1 mm, available
from Iiyama Precision Glass) made of synthetic quartz was placed in a
heat- and alkali-resisting rack and dipped in a cleaning solution for
ultrasonic cleaning, which was adjusted to have a predetermined
concentration. The glass substrate was kept dipped in the cleaning
solution for a night and cleaned by ultrasonic cleaning for 20 min. The
substrate was picked up, lightly rinsed with pure water, and cleaned by
ultrasonic cleaning in ultrapure water for 20 min. The substrate was
dipped in a 1N aqueous sodium hydroxide solution heated to 80° C.
for 10 min. Pure water cleaning and ultrapure water cleaning were
executed again. A quartz glass substrate for a DNA chip was thus
prepared.

[0064]4-2. Surface Treatment

[0065]A silane coupling agent KBM-603 (available from Shin-Etsu Silicone)
was dissolved in pure water at a concentration of 1% by weight (wt %) and
stirred at room temperature for 2 hrs. The cleaned glass substrate was
dipped in the aqueous solution of the silane coupling agent and left
stand still at room temperature for 20 min. The glass substrate was
picked up. The surface thereof was lightly rinsed with pure water and
dried by spraying nitrogen gas to both surfaces of the substrate. The
dried substrate was baked in an oven at 120° C. for 1 hr to
complete the coupling agent treatment, whereby an amino group was
introduced to the substrate surface. Next, N-maleimidocaproyloxy
succinimido (abbreviated as EMCS hereinafter) was dissolved in a 1:1
(volume ratio) solvent mixture of dimethyl sulfoxide and ethanol to
obtain an ultimate concentration of 0.3 mg/ml. As a result, an EMCS
solution was prepared. Here, EMCS is N-(6-maleimidocaproyloxy)succinimido
available from Dojindo.

[0066]The baked glass substrate was left stand and cooled and dipped in
the prepared EMCS solution at room temperature for 2 hrs. By this
treatment, the amino group introduced to the surface of the substrate by
the silane coupling agent reacted with the succinimide group in the EMCS
to introduce the maleimide group to the surface of the glass substrate.
The glass substrate picked up from the EMCS solution was cleaned by using
the above-described solvent mixture in which the EMCS was dissolved. The
glass substrate was further cleaned by ethanol and dried in a nitrogen
gas atmosphere.

[0067]4-3. Probe DNA

[0068]The microorganism detection probe prepared in the stage 1
(Preparation of Probe DNA) of Example 1 was dissolved in pure water. The
solution was dispensed such that the ultimate concentration (at ink
dissolution) became 10 μM. Then, the solution was freeze-dried to
remove water.

[0069]4-4. DNA Discharge by BJ Printer and Bonding to Substrate

[0070]An aqueous solution containing 7.5-wt % glycerin, 7.5-wt %
thiodiglycol, 7.5-wt % urea, and 1.0-wt % Acetylenol EH (available from
Kawaken Fine Chemicals) was prepared. The probe (Table 1) prepared in
advance was dissolved in the solvent mixture at a specific concentration.
An ink tank for an inkjet printer (trade name: BJF-850, available from
Canon) is filled with the resultant DNA solution and attached to the
printhead.

[0071]The inkjet printer used here was modified in advance to allow
printing on a flat plate. When a printing pattern is input in accordance
with a predetermined file creation method, about 5-picoliter of a DNA
solution can be spotted at a pitch of about 120 μm.

[0072]The printing operation was executed for one glass substrate by using
the modified inkjet printer to prepare an array. After confirming that
printing was reliably executed, the glass substrate was left stand still
in a humidified chamber for 30 min to make the maleimide group on the
glass substrate surface react with the thiol group at the nucleic acid
probe terminal.

[0073]4-5. Cleaning

[0074]After reaction for 30 min, the DNA solution remaining on the surface
was cleaned by using a 10-mM phosphate buffer (pH 7.0) containing 100-mM
NaCl, thereby obtaining a DNA chip in which single-stranded DNAs were
immobilized on the glass substrate surface.

[0075]5. Amplification and Labeling of Analyte

[0076]5-1. Amplification of Analyte: 1st PCR

[0077]The amplification reaction (1st PCR) and the labeling reaction (2nd
PCR) of a microbial gene to be provided as an analyte are shown in Table
4 below.

[0078]Amplification reaction of the reaction solution having the
above-mentioned composition was carried out using a commercially
available thermal cycler in accordance with the protocol illustrated in
FIG. 1. After the end of reaction, the primer was purified using a
purification column (QIAquick PCR Purification Kit available from
QIAGEN). Subsequently, the quantitative assay of the amplified product
was carried out.

[0079]5-2. Labeling Reaction: 2nd PCR

[0080]Amplification reaction of the reaction solution having the
composition shown in Table 5 was carried out using a commercially
available thermal cycler in accordance with the protocol illustrated in
FIG. 2.

[0081]After the end of reaction, the primer was purified using a
purification column (QIAquick PCR Purification Kit available from QIAGEN)
to obtain a labeled analyte.

[0082]6. Hybridization

[0083]Detection reaction was performed using the DNA chip prepared in the
stage 4 (Preparation of DNA Chip) and the labeled analyte prepared in the
stage 5 (Amplification and Labeling of Analyte).

[0084]6-1. Blocking of DNA Chip

[0085]Bovine serum albumin (BSA, Fraction V: available from Sigma) was
dissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt %
solution was obtained. Then, the DNA chip prepared in the stage 4
(Preparation of DNA Chip) was dipped in the solution at room temperature
for 2 hrs to execute blocking. After the end of blocking, the chip was
cleaned using a washing solution as described below, rinsed with pure
water and hydro-extracted by a spin dryer.

[0088]The hydro-extracted DNA chip was placed in a hybridization apparatus
(Hybridization Station available from Genomic Solutions Inc).
Hybridization reaction was carried out in a hybridization solution under
conditions as described below.

[0094]The DNA chip after the end of hybridization reaction was subjected
to fluorometry with a DNA chip fluorescent detector (GenePix 4000B
available from Axon). As a result, Propionibacterium (genus) was able to
be detected with a sufficient signal at a high reproducibility. The
results of fluorometry are shown in Table 6 below.

[0096]For proving the fact that the probe set shown in Table 1 can be
specifically hybridized only with Propionibacterium (genus), the result
of hybridization reaction with Escherichia coli (JCM 1649) is shown in
Table 7 below.

[0098]As is evident from the above description, a DNA chip was prepared
such that a probe, which was able to detect only Propionibacterium
(genus) in a specific manner, was immobilized. Further, the use of such a
DNA chip allowed the identification of an infectious disease pathogenic
bacterium, so the problems of the DNA probe derived from a microorganism
was able to be solved. In other words, the oligonucleotide probe can be
chemically produced in large amounts, while the purification or
concentration thereof can be controlled. In addition, for classification
of microbial species, a probe set capable of collectively detecting
bacterial strains of the same genus and differentially detecting them
from bacteria of other genera, was able to be provided.

[0099]Further, in addition to Escherichia coli as described above,
hybridization reaction was carried out on each of nucleic acids extracted
from the bacteria represented in the above-mentioned items (1) to (80).
The results thereof confirmed that no substantial reaction was observed
with respect to each of those bacteria in a manner similar to that of
Escherichia coli, except of Propionibacterium (genus).

[0100]The bacteria represented in the above-mentioned items (1) to (80)
are pathogenic bacteria for septicemia, and they cover almost all of the
pathogenic bacteria ever detected in human blood. Therefore, by using the
primer of the present embodiment, the nucleic acid of an infectious
disease pathogenic bacterium in blood can be extracted and then subjected
to hybridization reaction with the probe of the present invention,
whereby identification of Propionibacterium (genus) can be performed with
higher accuracy.

[0101]Further, according to the above-mentioned example, the presence of
an infectious disease pathogenic bacterium can be efficiently determined
with high accuracy by completely detecting the 16S rRNA gene from the
gene of the infectious disease pathogenic bacterium.

Example 2

Preparation of DNA Chip by which Various Bacterial Species can be
Simultaneously Determined

[0102]In a manner similar to the stage 1 (Preparation of Probe DNA) of
Example 1, probes having base sequences as shown in Table 8 below were
prepared.

[0103]Those probes are capable of specifically detecting certain bacterial
species (or genera) shown in the left column in the table just as one
specific to Propionibacterium (genus) of Example 1.

[0104]Further, those probes are designed such that they have the same Tm
value as that of a target, the same reactivity with a non-target
sequence, and the like so that the nucleic acid of the bacterial species
of interest can be specifically detected under the same reaction
conditions.

[0105]For the respective probes, probe solutions were prepared in a manner
similar to the stage 4-3 of Example 1. Subsequently, the inkjet printer
used in the stage 4-4 of Example 1 was employed to discharge each of the
probe solution on the same substrate to form a plurality of DNA chips
having spots of the respective probes being arranged at a pitch of about
120 μm.

[0106]One of the DNA chips was used for hybridization with the nucleic
acid extracted from Propionibacterium (genus) in a manner similar to the
stage 6 of Example 1. As a result, the spot of the probe which
specifically detected Propionibacterium (genus) showed almost the equal
fluorescence intensity as that of Example 1. In contrast, the spots of
other probes showed extremely low fluorescence intensity.

[0107]Further, other prepared DNA chips were used for hybridization with
the bacteria listed in Table 8 except of Propionibacterium (genus). As a
result, the spot of Propionibacterium (genus) showed extremely low
fluorescence intensity, while the spot of the probe for the bacterial
species of interest showed extremely high fluorescence intensity.
Therefore, the DNA chip prepared in the present example was confirmed
that it was able to simultaneously determine 16 bacterial species and 6
genera listed in Table 8 in addition to Propionibacterium (genus). By
simultaneously using probes for a target species and the corresponding
genus (for example, Propionibacterium (genus) and Propionibactrium
acnes), highly accurate identification of the target species or
simultaneous identification of a plurality of target species of the same
genus can be performed.

Example 3

[0108]Using the DNA chip prepared in Example 2, detection was attempted
when a plurality of bacterial species was present in an analyte.

[0109]A culture medium in which Propionibacterium acnes as
Propionibacterium (genus) and Eggerthella lenta were cultured was
prepared and subjected to the same treatment as that of Example 1 to
react with the DNA chip.

[0110]As a result, only the spots of the probes having SEQ ID NOS. 49, 50,
51, 87, 88, 89, and 94 showed high fluorescence intensity, so the
coexistence of those bacteria was able to be simultaneously confirmed.

[0111]The present invention is not limited to the above-mentioned
embodiments and various changes and modifications can be made within the
spirit and scope of the present invention. Therefore to apprise the
public of the scope of the present invention, the following claims are
made.

[0112]While the present invention has been described with reference to
exemplary embodiments, it is to be understood that the invention is not
limited to the disclosed exemplary embodiments. The scope of the
following claims is to be accorded the broadest interpretation so as to
encompass all such modifications and equivalent structures and functions.

[0113]This application claims the benefit of Japanese Patent Application
No. 2006-306004, filed Nov. 10, 2007, which is hereby incorporated by
reference in its entirety.